For ROS detection in BALF, a 2′, 7′-dichlorodihydrofluorescein (DCF) ROS/RNS assay kit (ab238535, Abcam) was performed according to the protocol of manufacturer. For intracellular ROS levels, NCI-H292 cells (CRL-1848, ATCC, Manassas, VA, USA) were incubated in a humidified chamber at 37 °C with 5% CO2 for 45 min with a 2′, 7′-dichlorodihydrofluorescein diacetate. Fluorescence was determined at 485 nm excitation/535 nm emission.
Nci h292 cells
Manufactured by American Type Culture Collection
Sourced in United States
NCI-H292 cells are a human pulmonary mucoepidermoid carcinoma cell line derived from the lung of a 42-year-old male. These cells are widely used in research related to respiratory and lung health.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Athymic nu nu, by Charles River Laboratories (2 mentions)
Nci h292 human lung cancer cells, by American Type Culture Collection (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Dcf ros rns assay kit, by Abcam (1 mentions)
Iscove modified dulbecco media (imdm), by Lonza (1 mentions)
A549 cell, by American Type Culture Collection (1 mentions)
Dhbe copd, by Lonza (1 mentions)
Flag peptide, by Merck Group (1 mentions)
Anti flag m2 magnetic bead, by Merck Group (1 mentions)
24 well plate, by Corning (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Dcp bio1, by Kerafast (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Coomassie bradford protein assay kit, by Thermo Fisher Scientific (1 mentions)
Lapatinib, by LC Laboratories (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
32 protocols using nci h292 cells
1
Quantifying ROS/RNS in BALF and Cells
2
NCI-H292 Cell Viability Assay
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Human airway epithelial NCI-H292 cells (ATCC, Manassas, VA, USA) were cultured in RPMI 1640 medium supplemented with 200 mM l -glutamine, 10% (vol/vol) fetal calf serum, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 2.5 mg/liter glucose and buffered with 25 mM HEPES at 37°C in a humidified, 5% CO2 water-jacketed incubator. Then, the cells were cultured overnight under serum-free conditions and treated with the peptides (5 or 20 μM). After 24 h, the cell viability was assessed by measuring the release of lactate dehydrogenase (LDH) as previously reported (22 (link)).
3
NCI-H292 Xenograft Mouse Model
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All animal experiments were approved by our Institutional Animal Care Committee (IACUC). Mice were maintained with free access to standard chow and water. NCI-H292 human lung cancer cells were obtained from the American Type Culture Collection (ATCC). They were cultured in RPMI medium supplemented with 10% (v/v) fetal bovine serum (FBS). The cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C. For H292 xenograft studies, 7- to 9-week-old female athymic nu/nu (Charles River Laboratories) mice were inoculated subcutaneously in the right hind flank with 5 × 106 NCI-H292 cells (ATCC) suspended 1:1 (v/v) with Matrigel in serum-free medium. Clinical observations, body weights, and digital caliper tumor volume measurements were made two times weekly once tumors became measureable. Tumor volumes were calculated with the formula (ab2)/2, where a is the longer and b is the smaller of two perpendicular diameters.
4
Propagation of Influenza and RSV Viruses
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Influenza, strain A PR/8/34, and respiratory syncytial virus (RSV)‐A2 were used. RSV was propagated in HEp‐2 cells in IMDM (Lonza) culture medium supplemented with 1% FCS and influenza on NCI‐H292 cells (ATCC CRL 1848) in RPMI‐1640 with 1% FCS. At day 3 postinfection, when cytopathic effects were observed, the supernatant was harvested. Cell debris was removed by centrifugation at 3000 g for 10 minutes and the supernatant was snap frozen and stored at −80°C.
5
Influenza Virus Infection Assay
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Virus stocks of influenza A/H1N1 subtype: FM/1/47, Bangkok/10/83, Beijing/262/95, Oita/OU1P3-3/09, and A/H3N2 subtype: Udorn/72, Aichi/2/68, and B/Ibaragi/2/85 were used for virus infection. All strains were grown in the chorioallantonic fluid of 10-day-old chicken eggs. The aliquots of each virus preparation were stored at −80 °C until use. Human lung carcinoma, NCI-H292 cells (ATCC CRL1848) or A549 cells (ATCC CCL185) were used for the host cells. These cells were grown in RPMI-1640 supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin (GIBCO, NY, USA). Influenza HA vaccine “SEIKEN” containing a mixture of A/California/7/2009 (H1N1), A/Victoria/210/2009 (H3N2), and B/Brisbane/60/2008 was used as HA protein preparation (Denka-Seiken, Tokyo, Japan).
6
Influenza A virus propagation in eggs
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Viral stocks of influenza A/H1N1/Oita/OU1P3-3/09 and A/H3N2/Udorn/72 were used for viral infection. Viruses were grown in the chorioallantoic fluid of 10-day-old chicken eggs. Aliquots of each viral preparation were stored at −80 °C until use. Human lung carcinoma NCI-H292 cells (ATCC CRL1848) were used as host cells. The cells were grown in RPMI-1640 supplemented with 10% fetal bovine serum, 100 IU ml−1 penicillin, and 100 μg ml−1 streptomycin (Gibco).
7
NCI-H292 Xenograft Mouse Model
8
Anti-influenza Virus Activity Assay
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Anti-influenza virus activity was determined according to the method described previously [12 (link),13 (link)]. Human NCI-H292 cells (ATCC CRL1848) grown in a 48-well plate were infected with influenza virus A/H3N2/Udorn/72 strain at a multiplicity of infection (moi) of 2.5. Various concentrations of the lectin were added simultaneously into the cell cultures. At 24 h post-infection (hpi), the infected cells were fixed with 80% acetone and stained with 0.5% amide black in 45% ethanol and 10% acetic acid. The stained plates were pictured with a grayscale. The color densities of the pictures were quantitated by densitometry with the NIH-ImageJ 1.48v software. The infected cell cultures, in the absence of lectin, exhibited severe cytopathic effects, and almost all cells on the wells were gone, with the percentage of cell viability shown as 0%. On the other hand, cells in the mock-infected cell cultures were intact, in which the percentage of cell viability was shown as 100%.
9
Cytokine-Induced Airway Cell Response
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Human recombinant IL-4 was provided by InvivoGen (California, United States) and added to the cell culture media at a final concentration of 40ng/mL. NCI-H292 cells were obtained from ATCC (Virginia, United States). Primary cells (NHBE, DHBE-Asthma and DHBE-COPD) were obtained from Lonza (Basel, Switzerland). More detailed information about the individuals from which the primary cells were derived is listed in Table 1. All PCR and sequencing primers were obtained from Microsynth (Switzerland).
10
Airway Epithelial Cell Culture Protocol
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Human bronchial epithelial (HBE) cells and human NCI-H292 cells (lung mucoepidermoid carcinoma cell line) were purchased from the ATCC (Manassas, VA), propagated and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum in a 37 C, 5% CO 2 incubator. After reaching >90% confluence, the cells were incubated in serum-free medium for 12 hours to synchronize the cell cycle and to return serum-activated signaling pathways to basal levels before exposure to CS extract (CSE).
To isolate and identify airway epithelial cell-specific response in vitro, primary mouse tracheal epithelial cells (MTECs) were obtained from the tracheas of C57BL/6 mice or Atf3 À/À mice. Harvesting of MTECs was performed in accordance with previously described methods, 17 (link) and the cells were seeded onto Transwell inserts in 12-well plates (0.4-mm pore, 12-mm diameter; Corning, Corning, NY) and cultured at 37 C in a 5% CO 2 incubator. After an approximately 2-week proliferative phase, the cells reached confluence. The apical medium was removed to switch the cells to the differentiation phase (an additional 2 weeks) at an air-liquid interface (ALI) to achieve mucociliary differentiation. The cells were then exposed to CSE after differentiation was established.
To isolate and identify airway epithelial cell-specific response in vitro, primary mouse tracheal epithelial cells (MTECs) were obtained from the tracheas of C57BL/6 mice or Atf3 À/À mice. Harvesting of MTECs was performed in accordance with previously described methods, 17 (link) and the cells were seeded onto Transwell inserts in 12-well plates (0.4-mm pore, 12-mm diameter; Corning, Corning, NY) and cultured at 37 C in a 5% CO 2 incubator. After an approximately 2-week proliferative phase, the cells reached confluence. The apical medium was removed to switch the cells to the differentiation phase (an additional 2 weeks) at an air-liquid interface (ALI) to achieve mucociliary differentiation. The cells were then exposed to CSE after differentiation was established.
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